Extraction of natural ferulate and coumarate from biomass

ABSTRACT

A process for a reactive separation of organic molecules from biomass includes a reaction step for the biomass, a simultaneous extraction step using a solvent, and a filtration step to recover products, wherein the products comprise ferulic acid and/or coumaric acid. The products are extracted from the biomass in a pressurized stirred batch reactor using a liquid extraction solvent and a base in which the ferulate and the coumarate remain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation and claims priority to U.S. patentapplication Ser. No. 16/606,643, filed on Oct. 18, 2019, and entitled,“EXTRACTION OF NATURAL FERULATE AND COUMARATE FROM BIOMASS,” which is afiling under 35 U.S.C. 371 as the National Stage of InternationalApplication No. PCT/US2018/028566, filed on Apr. 20, 2018, entitled,“EXTRACTION OF NATURAL FERULATE AND COUMARATE FROM BIOMASS,” whichclaims the benefit of and claims priority to U.S. ProvisionalApplication No. 62/487,911, filed on Apr. 20, 2017 and entitled“Extraction of Natural Ferulate and Coumarate from Biomass”, all ofwhich are incorporated herein by reference in their entirety for allpurposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under grant number1647923 awarded by the U.S. National Science Foundation Phase I SBIR.The government has certain rights in the invention.

REFERENCE TO A MICROFICHE APPENDIX

Not applicable.

BACKGROUND

Ferulic acid (3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid,3-(4-hydroxy-3-methoxyphenyl)acrylic acid) is a powerful anti-oxidantused in consumer products and pharmaceuticals. The flavor and fragranceindustry transforms ferulic acid to vanillin using enzymatic processes[see for example, I. Labuda, et al. (Kraft Foods Inc) U.S. Pat. No.5,128,253A (1991); A. Muheim, et al. (Givaudan Roure (International)SA), U.S. Pat. No. 6,235,507 B1 (1997)]. Natural vanillin produced fromnatural ferulic acid is of particular importance due to the volatility,high cost, and scarcity of natural vanilla extract derived from vanillabeans.

SUMMARY

In an embodiment, a process for a reactive separation of organicmolecules from biomass comprises a reaction step for the biomass, asimultaneous extraction step using a solvent, and a filtration step torecover products, wherein the products comprise a ferulate or acoumarate. The products are extracted from the biomass in a pressurizedstirred batch reactor using a liquid extraction solvent and a base inwhich the ferulate and the coumarate remain.

In an embodiment, a reactive separation process for a separation oforganic molecules including acidic esters, terpenoids, sterols,carbohydrates, and flavonoids from biomass, the process comprises areaction step using a base in contact with the biomass, a simultaneoussolvent extraction step using a solvent, and a filtration step torecover products comprising the organic molecules.

In an embodiment, a process to extract a ferulate and a coumarate fromagricultural biomass in a packed bed reactor comprises contacting thebiomass with a solvent and a base in the packed bed reactor, wherein theagricultural biomass acts as a stationary bed.

These and other features will be more clearly understood from thefollowing detailed description taken in conjunction with theaccompanying drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present disclosure and theadvantages thereof, reference is now made to the following briefdescription, taken in connection with the accompanying drawings anddetailed description.

FIG. 1 is a schematic process illustration of a stirred batch reactoraccording to an embodiment.

FIG. 2 is a schematic process illustration of a continuous extractionprocess using a packed bed reactor according to an embodiment.

FIG. 3 illustrates the chemical structure or ferulic acid, variousferulates, coumaric acid, and various coumarates.

FIG. 4 illustrates the results of an HPLC/UV-vis chromatogram.

FIG. 5 illustrates the results of an HPLC/UV-vis chromatogram for theoil of Example 1.

FIG. 6 illustrates the results of an HPLC/UV-vis chromatogram for theoil of Example 2.

FIG. 7 illustrates the results of an HPLC/UV-vis chromatogram for theoil of Example 3.

FIG. 8 illustrates the results of an HPLC/UV-vis chromatogram for theoil of Example 4.

FIG. 9 illustrates the results of an HPLC/UV-vis chromatogram for theoil of Example 5.

FIG. 10 illustrates the results of an HPLC/UV-vis chromatogram for theoil of example 6.

FIG. 11 illustrates the results of the yield of methyl ferulate on a drymass basis per hour of Example 6.

FIG. 12 illustrates the results of an HPLC/UV-vis chromatogram for theoil of Example 7.

FIG. 13 illustrates the results of an HPLC/UV-vis chromatogram for acrude oil from Example 8 containing ethyl ferulate and ethyl coumarateand hydrolyzed crude oil.

FIG. 14 illustrates the results of an HPLC/UV-vis chromatogram for theaqueous phase of Example 9 following corn oil removal.

FIG. 15 illustrates the results of ¹HNMR spectroscopy of the corn oilisolated in Example 9.

FIG. 16 illustrates the results of an HPLC/UV-vis chromatogram for thesolution of Example 10 following hydrolysis.

FIG. 17 illustrates the results of an HPLC/UV-vis chromatogram forExample 13 showing ferulic acid with a purity of greater than or equalto 98% pure.

DETAILED DESCRIPTION

Ferulic and coumaric acid are powerful anti-oxidants. Ferulic acid isalso used in the flavor and fragrance industry to produce naturalvanillin. Disclosed herein a process for the extraction of ferulate(ferulic acid ester) and coumarate (coumaric acid ester) fromagricultural biomass containing ferulic and coumaric acid linkages isdescribed. The extracted ferulate and coumarate can be classified asnatural, for example following European 1334/2008 and US Food and Drugadministration (FDA) 21CFR101.22 regulations regarding natural labeling.The extracted ferulate can be hydrolyzed to ferulic acid in theextraction mixture, or hydrolyzed after separation. Additional cinnamicacids, sugars, corn oil and fatty acids are also extracted during theprocess.

Consumer demands for natural products and ingredients have driven theindustry to seek new sources of natural vanilla, while vanilla derivedfrom synthetic, non-natural sources such as petrochemicals and eugenolhave seen a decrease in demand. Carbon 13 NMR of vanillin is used todetermine the source of vanillin by the ratio of ¹³C and ¹²C at theeight carbons of vanillin, with the carbons of the aldehyde and themethoxy group determined to be the most important [G. Remaud, et al. (J.Ag. Food Chem.) 45 (1997); E. Tenailleau, et al. (J. Ag. Food. Chem.) 52(2004)]. Synthetic sources such as guaiacol can be discriminated fromnatural vanillin sourced from beans and ferulic acid, with thresholdsdependent on the experimental setup.

Much of the supply of natural ferulic acid is extracted during theprocessing of rice bran oil [see, for example, H. Taniguchi, et al.(Wakayama, J P) U.S. Pat. No. 5,288,902 (1994); Z. Sun, et al. (ZhejiangHangzhou Xinfu Pharmaceutical Co., Ltd) U.S. Pat. No. 7,462,470 (2006);CN 103553903A; CN 103553902A; CN 103254993B]. Ferulic acid is alsocontained in agricultural biomass such as miscanthus, corn byproducts(fiber, bran, stover, fines, gluten feed, etc.), rice, wheat, beets,beet fiber, beet pulp, and other crops. Typical methods for extractingferulic acid include alkali extraction [R. Antrim, et al. (StandardBrands Incorporated), U.S. Pat. No. 4,038,481 (1977); A. Muir, Westcott,N. (CA Minister Agriculture & Food) EP 0817824B1 (2001); CN 103254064B(2015); CN 103319328B (2015)] and enzymatic processes [D. Wong, et al.(US Department of Agriculture), U.S. Pat. No. 8,361,764 B1 (2013); C.Buchanan, et al. (Eastman Chemical Co), U.S. Pat. No. 6,352,845 B1(2002)]. Alternatively, ferulic acid is extracted as several ferulicacid phytosterol esters such or γ-oryzanol [C. Abbas, et al. (ArcherDaniels Midland Co) US 20030235633 A1 (2003); C. Abbas, et al. (ArcherDaniels Midland Co) U.S. Pat. No. 7,833,994B2 (2010); R. Moreau, et al.(US Department of Agriculture) U.S. Pat. No. 5,843,499A (1998); P. Das,et al. (Council of Scientific and Industrial Research) U.S. Pat. No.5,869,708A (1999)]. Excess alcohol extraction has been used to removelignin from biomass [V. Diebold, et al. (Alcell Technologies Inc.) U.S.Pat. No. 4,100,016A (1978)], and similar extractions have been used toextract ferulic acid esters. For example, in the presence of methanol,the extract contains methyl ferulate (methyl3-(4-hydroxy-3-methoxyphenyl)acrylate), while in the presence of ethanolthe extract contains ethyl ferulate (ethyl3-(4-hydroxy-3-methoxyphenyl)acrylate). Naturally derived ferulate canbe transformed into natural ferulic acid via alkaline hydrolysis.

This application describes a process for extracting ferulate (ferulicacid esters of the variety methyl-, ethyl-, propyl-, butyl-, or anyvariation thereof) and coumarate from agricultural biomass such asmiscanthus and corn byproducts. The extraction can be carried out ineither batch or continuous operation. Under conditions compatible withcooking procedures, the extracted ferulic acid can be marketed asnatural in the European Union and the United States. For example, theextracted products can be marked as natural following European 1334/2008and/or US Food and Drug administration (FDA) 21CFR101.22 regulationsregarding natural labeling. Ferulate can be hydrolyzed to ferulic acid.

As disclosed herein, a process for extracting ferulate and coumarate caninclude a reaction step and/or extraction step for the extraction offerulates and coumarates from biomass. A filtration step can be used torecover the products. The reaction step, which can occur prior to orsimultaneously with the extraction step, can occur with a base. Theextraction step can occur in the presence of a solvent. The products cancomprise at least one of: ferulate (ferulic acid ester), and coumarate(coumaric acid ester).

The present process is related to the extraction of ferulates (ferulicacid esters) and optionally coumarates from agricultural biomass. Thestructures of exemplary ferulates and coumarates are shown in FIG. 3. Asshown in FIG. 3(A), R can represent a hydrogen or an alkyl group suchthat: ferulic acid: R═H, methyl ferulate: R═CH₃, ethyl ferulate: R═C₂H₅,propyl ferulate: R═C₃H₇, butyl ferulate: R═C₄H₉. As shown in FIG. 3(B) Rcan represent a hydrogen or an alkyl group such that: coumaric acid:R═H, methyl coumarate: R═CH₃, ethyl coumarate: R═C₂H₅, propyl coumarate:R═C₃H₇, butyl coumarate: R═C₄H₉.

In the present application, any suitable biomass can be used as astarting stock. Various exemplary biomass feedstocks can include, butare not limited to, miscanthus, corn bran, corn fiber, corn gluten feed,distillers' grain, corn stover, corn gluten meal, beet fiber, ricehulls, and/or other agricultural residues.

The process can include an extraction step using a solvent. In someembodiments, the solvent can comprise an alcohol (e.g., an organicalcohol) along with a co-solvent such as water. While not wishing to belimited by theory, it has been noted that the identity of the ferulateis directly linked to the chosen alcohol in the solvent. For example,the use of methanol yields methyl ferulate and the use of ethanol yieldsethyl ferulate. Esters of coumaric acid are simultaneously extracted. Insome embodiments, the extraction solvent can comprise 0-50% water and apure aliphatic alcohol or mixtures of aliphatic alcohols (e.g.,methanol, ethanol, n-propanol, iso-propylalcohol, n-butanol, 2-butanol,tert-butanol, n-pentanol, etc.), where the aliphatic alcohol cancomprise 50-100% of the solvent. In some embodiments, the solvent mayinclude only water and one or more aliphatic alcohols. In still otherembodiments, water may not be present in the solvent, and rather 100%alcohol of the variety methanol or ethanol can be used for the solvent.When ethanol is biologically obtained (e.g., produced from fermentation,etc.), ethyl ferulate can be labeled as natural when using such ethanolas the solvent in the extraction. In some instances, the products can beconsidered natural when all of the carbons in the products (e.g., in theethyl ferulate, etc.) are naturally sourced.

In some embodiments, a reaction step can also be carried out using abase. This reaction step can be carried out at the same time as theextraction. For example, base can be added to the solvent to enhancesolubility and rate of extraction at lower reaction temperatures. Basesinclude any first or second group hydroxides such as sodium hydroxide orpotassium hydroxide, carbonates, bicarbonates, and ammonium inconcentrations of about 0 to about 1 N (molar equivalents of base perliter of solvent). For example, the concentration of the base can bebetween 0.01 and about 0.1 N, or between about 0.02 and about 0.06 N, orabout 0.04 N.

Various process designs can be used to carry out the extraction and/orreaction steps. Any suitable reactor configuration capable of contactingthe biomass feedstock with the solvent and/or base can be used. Forexample, a batch reactor and/or a continuous flow reactor can be used.FIG. 1 illustrates a process 100 for the extraction of ferulates and/orcoumarates from biomass using a batch reactor such as a stirred batchreactor 102. Under stirred batch reaction conditions, the startingmaterials (e.g., the solvent including an alcohol with or without water,optionally a base, and the biomass) can be mixed together in a stirredbatch reactor 102 and sealed shut. Alternatively, the biomass,solvent(s), and base can be pre-mixed as a slurry and fed into thereactor 102 using a pump or gravity. The mass to mass ratio forsolvent:biomass can be in the range of about 4 to about 20. In someembodiments, the mass to mass ratio for solvent:biomass of about 10 toabout 15 can be used. The reaction atmosphere can be purged of oxygenusing an inert gas from an inert gas source 104 such that the atmosphereis inert with argon, helium, nitrogen, or a mixture thereof. Hydrogen(0-100%) atmospheres give similar results to a purely inert atmosphere.The purge gas can also be used to pressurize the reactor 102. Thereactor 102 can be pressurized to between about 1 to about 3 bar at roomtemperature, with about 1 bar preferred. The reactor 102 can be heatedat 100-300° C. hr⁻¹ (e.g., between about 200° C. hr⁻¹ to about 300° C.hr⁻¹, or about 300° C. hr⁻¹) to a dwell temperature of between about 80°C. and about 250° C., or between about 100° C. to 250° C. The reactor102 can maintain the maximum extraction temperature for between 1-15hours, for example for about 12 hours. The stirring mechanism can beoperated from about 100 to about 600 rpm, for example, about 200 rpm.

After reaction, the reactor 102 can be cooled to room temperature. Thereactor 102 can be purged to atmospheric pressure. The solid/liquid postreaction slurry 106 can be removed from the reactor 102 either by pumpor by gravity and filtered for solids. The solid residue can be rinsedwith clean solvent 108 using 50%-150% the volume of the filtered solids.The wet solids can then be pressed to further remove liquid captured inthe solids. The resulting products 110 can then be captured for furtherprocessing.

FIG. 2 illustrates a continuous extraction reactor scheme 200 for theextraction and/or reaction of the biomass to remove ferulates and/orcoumarates. As shown in FIG. 2, a packed bed reactor 202 can be used tocontinuously extract ferulate from agricultural biomass 204. In thisconfiguration, the agricultural biomass bed 204 acts as the stationarybed. Heated and pressurized solvent 206 can be pumped over the biomassbed 204, which extracts ferulate and coumarate. A back pressureregulator 208 downstream from the packed bed maintains the reactor 202at a steady pressure, typically between about 1 to about 30 bar. Thetypical operating conditions can include a bed temperature of betweenabout 80° C. and about 250° C., a reactor pressure of between about 1and about 30 bar. The solvent flow rate may be suitable for the reactorconditions and can have an liquid hourly space velocity of about 0.06hr⁻¹ to 0.6 hr⁻¹. The reactor 202 could be operated such that theextraction solvent either enters the biomass bed 204 a single time or isrecycled through the reactor several times. For example, optionalrecycle line 210 can be used to recycle the solvent for recycle throughthe packed biomass bed 204. The extraction solvent 206 can be collectedin a reservoir 212 at the exit of the reactor 202.

During operation, the reactor 202 can be purged and pressurized with aninert gas from an inert gas source 214. The temperature of the reactor202 can then be ramped up at a rate of between about 100 and about 300°C. hr⁻¹ to the desired operating temperature. After reaching reactiontemperature, the reactor 202 can be operated for between about 2-8hours. The reactor 202 can then be cooled and the flow of solventbriefly increased to wash the biomass bed 204. The solvent flow can bestopped after the reactor temperature decreases to approximately 20-40°C. The pressure can be returned to atmospheric pressure after thereactor 202 reaches room temperature. The spent biomass can then beemptied from the reactor 202.

The resulting products can be characterized through testing. Forexample, the liquid product stream can be analyzed with a HPLC equippedwith a UV-vis detection. The column is a Zorbax SB-Phenyl reversed-phaseC18 HPLC column. Two methods can be used to quantify the mass offerulate, coumarate, coumaric acid, and ferulic acid in the liquidproduct. In Method 1, a gradient of water and acetonitrile can be usedto elute the products at 30° C. and a flow rate of 0.5 mL min⁻¹. InMethod 2, a gradient of 1 mM of aqueous trifluoroacetic acid andacetonitrile elute the products at 30° C. and a flow rate of 1.0 mLmin⁻¹. Ferulate and coumarate can be quantified by Method 1 or 2.Ferulic acid and coumaric acid can only be quantified by Method 2,although ferulic acid and coumaric acid are visible using Method 1, thepeaks, however, are not well resolved. FIG. 4 shows a HPLC chromatogramfor chemical standards of ferulic acid, coumaric acid, methyl ferulate,methyl coumarate, and ethyl ferulate. As shown in FIG. 4, methanol wasinjected which contained 1.02, 1.00, 1.07, 1.08, and 1.03 mM of coumaricacid (10.0 min), ferulic acid (12.1 min), methyl coumarate (26.1 min),methyl ferulate (26.8 min), and ethyl ferulate (30.4 min), respectively.Standard curves for each ferulate and coumarate were created todetermine the concentration and yield of each product. The concentrationof ferulate and ferulic acid was determined at a wavelength of 238.8 nmwhile coumarate and coumaric acid were quantified at 310.8 nm. Testscarried out as described herein on products from exemplary reactor runswas used to calculate the total yield (based on the starting mass of drybiomass) of ferulate, and the total yield from each biomass was near thetheoretical content as shown in Table 1.

TABLE 1 Theoretical and extracted ferulate content of agriculturalbiomass Theoretical ferulic acid content Extracted ferulate contentBiomass (g_(ferulic acid)/g_(biomass)) (g_(ferulate)/g_(biomass)) Cornbran 2.8-3.1 ¹ 2.6 Corn fiber 1.0-1.8 ¹ 1.6 Corn gluten feed 1.0-1.8 ¹0.8 Corn stover 0.4 Miscanthus 0.5-1.0 ² 0.6

A number of additional processing steps can be carried out on the liquidproducts from the extraction and/or reaction steps. Initially, theferulate can be concentrated and an oil phase referred to as “corn oil”can be removed. The ferulate in the resulting solution, or alternativelyin the liquid products prior to the concentration to the liquidproducts, can be hydrolyzed to ferulic acid. Lignin and polysaccharidesin the resulting liquid phase can be precipitated to further purify theferulic acid. An organic solvent can then be used to extract the ferulicacid followed by hot filtration and precipitation of a solid ferulicacid product. Additional purification steps can also be carried out tofurther purify the ferulic acid as desired.

In an embodiment, the ferulate in the liquid product from the extractionand/or reaction steps can be concentrated. Following extraction offerulate into the basic alcohol solution, an evaporator (e.g., a rotaryevaporator, etc.) can be used to concentrate the alcohol/ferulatesolution until the alcohol concentration is between about 0% and about75% of the total solution by mass, or between about 40% and about 50%alcohol by mass. Concentration of the alcohol/ferulate solution canresult in a viscous brown oil. Liquid extraction can be used to remove avariety of fatty acid esters comprised of primarily esters of oleic andlinoleic acids and referred to herein as the corn oil from thealcohol/ferulate solution. While not wishing to be limited by theory, ithas been noted that the identity of the esters of oleic and linoleicacids are directly linked to the chosen alcohol in the solvent. Forexample, the use of ethanol yields the ethyl esters of oleic andlinoleic acids and use of methanol yields the methyl esters of oleic andlinoleic acid. Prior to liquid extraction of the corn oil, theconcentrated alcohol/ferulate solution can be diluted with a solution ofaqueous base. For example, between about 50-300 g aqueous base solutioncan be added per 100 g concentrated alcohol/ferulate solution. Bases caninclude any first or second group hydroxides such as sodium hydroxide orpotassium hydroxide, carbonates, bicarbonates, and ammonium. In someembodiments, sodium hydroxide can be used in concentrations between0.1-10 N. Following dilution of the concentrated alcohol/ferulatesolution with aqueous base, corn oil is removed by a liquid-liquidextraction with an organic solvent including, but not limited to,pentane, hexane, heptane, cyclohexane, benzene, toluene, diethyl ether,or mixtures thereof. In some embodiments, the organic solvent used inthe liquid-liquid extraction is hexane. Corn oil can be recovered byevaporation of the organic solvent used for the liquid-liquidextraction. Extraction of corn oil from the ferulate solution can becompleted before and/or after hydrolysis of ferulate to ferulic acid.Extraction of corn oil before hydrolysis can be used to advantageouslyreduce the processing volume of the hydrolysis step. The extracted cornoil can have a phosphorous content of less than 250 ppm, less than 150ppm, less than 100 ppm, less than 50 ppm, or less than 25 ppm, all bymass. In some embodiments, the extracted corn oil can contain less than3 ppm by mass phosphorous. The extracted corn oil can have a free fattyacid content (as oleic acid) of less than about 3% by mass of the oil.In some embodiments the extracted corn oil can have a free fatty acidcontent (as oleic acid) of less than about 1.5% by mass of the oil.

The liquid solution containing the ferulate can be hydrolyzed before orafter removal of the corn oil from the products. The liquid solution canbe hydrolyzed under basic conditions to convert at least a portion ofthe ferulate to ferulic acid. In this process, additional base can beadded to the ferulate solution and the solution can then be heated above20° C. to initialize the hydrolysis. Bases include any first or secondgroup hydroxides such as sodium hydroxide or potassium hydroxide,carbonates, bicarbonates, ammonium, and any combination thereof. Thebase used can be the same or different than the base used in the initialreaction step with the biomass. In some embodiments, sodium hydroxidecan be used as the base, and the sodium hydroxide can be used inconcentrations between 0.1-10 N. The mixture can be heated to betweenabout 30-100° C. for about 0.5-5 hours. Under these conditions, ethyl-and methyl ferulate can hydrolyze (e.g., partially hydrolyze,substantially completely hydrolyze, or completely hydrolyze) to ferulicacid.

Once hydrolyzed, the resulting mixture containing the ferulic acid canbe treated to precipitate byproducts of the extraction process such asany lignin and polysaccharides. The precipitation process can be carriedout by acidifying the ferulic acid containing solution to selectivelyprecipitate lignin and polysaccharides while the ferulic acid remains insolution. The concentration of ferulic acid can be adjusted to betweenabout 1-20 g/L by addition of water to the ferulic acid containingsolution, and the solution can be maintained at a temperature of betweenabout 20-100° C. The ferulic acid solution can then be acidified to a pHof between about 3-6 by addition of an acid. Suitable acids useful inthe acidification of the ferulic acid solution can include, but are notlimited to, hydrochloric acid, nitric acid, phosphoric acid, sulfuricacid, acetic acid, and combinations thereof. In some embodiments theacid can be sulfuric acid. Upon acidification, the by-products canprecipitate in the acidified solution. The acidified solution can befiltered to remove the precipitated byproducts including lignin andpolysaccharides which precipitate as solid materials. In someembodiments, a powder such as diatomaceous earth, celite, alumina,and/or silica may be added to the ferulic acid solution beforeacidification. Upon acidification, precipitated byproducts such aslignin and polysaccharides can bind to the inert powder, increasing theease of filtration and preventing fouling. In some embodiments,centrifugation can be used to isolate the solid precipitated lignin andpolysaccharides from the acidified solution in place of filtration.

The acidified solution having at least a portion of the byproductsremoved can be further purified using a variety of processing steps. Insome embodiments, the ferulic acid can be further purified using anextraction process using an organic solvent. In this step of apurification process, a liquid-liquid extraction can be used to extractferulic acid from an aqueous solution to an organic solvent. Suitableorganic solvents may include, but are not limited to, ethyl acetate,diethyl ether, dichloromethane, hexane, heptane, pentane, toluene,xylenes, and mixtures thereof. In some embodiments, the organic solventcan be or include ethyl acetate. The aqueous solution containing ferulicacid can be further acidified to a pH of between about 1-5 by additionof an acid before extraction with an organic solvent. Suitable acids caninclude, but are not limited to, hydrochloric acid, nitric acid,phosphoric acid, sulfuric acid, acetic acid, and combinations thereof.In some embodiments, the acid can be or include sulfuric acid. Uponacidification, the aqueous ferulic acid solution can be extracted withthe organic solvent using liquid-liquid contact. Typically the volume oforganic solvent used to extract the ferulic acid is between about 0.5-3times the volume of the aqueous ferulic acid solution. Followingextraction of the ferulic acid into the organic solvent, the organicsolvent phase can separated from the aqueous phase, and the organicsolvent can then be removed by evaporation (e.g., rotary evaporation,etc.) to yield solid or semi-solid ferulic acid. In some embodiments,the solid or semi-solid ferulic acid can have a purity of between about10-50% or between about 20-40% by mass. The resulting ferulic acid canbe used as product or subjected to further purification depending on theuse and product purity requirements.

The solid or semi-solid ferulic acid can be further purified using adissolution-precipitation process. In some embodiments, the solidferulic acid of low purity (e.g., between about 10% to about 50% orbetween about 20-40% purity by mass) can be further purified through ahot filtration and precipitation process. In this process, the solidferulic acid can be dissolved in water at a concentration of 1-10 g/Lferulic acid and heated to between about 60-100° C. The heated mixturecan be filtered to remove non-soluble materials while maintaining atemperature of between about 60-100° C. The filtrate can then be cooledto precipitate solid ferulic acid at a purity greater than the startingpurity. In some embodiments, the dissolution-precipitation process canincrease the purity of the solid ferulic acid to between about 60-99%purity by mass. Alternatively, the concentration of ferulic acid in thefiltrate can be increased from 1-10 g/L to 10-35 g/L by evaporating aportion of the aqueous filtrate prior to cooling to precipitate solidferulic acid with a purity of between about 60-99% by mass.

Additional purifications processes can be used to purify the solid orsemi-solid ferulic acid in addition to the dissolution-precipitationprocess or in place of the dissolution-precipitation process. In someembodiments, liquid chromatography can be used increase the purity offerulic acid. In this process, liquid chromatography can be used toincrease the purity of ferulic acid from between about 10-50% purity orbetween about 20-40% purity to about 99% or greater purity if appliedbefore a dissolution-precipitation process. If applied after adissolution-precipitation process, liquid chromatography can be used toincrease the purity of ferulic acid from about 60-99% purity by massto >99% purity by mass. Liquid chromatography techniques used in thisstep include but are not limited to: ion exchange chromatography andsize exclusion chromatography.

Once purified, the resulting product can include a concentrated ferulicacid solution or solid or semi-solid ferulic acid. The products can thenbe used for a variety of commercial uses.

EXAMPLES

The disclosure having been generally described, the following examplesare given as particular embodiments of the disclosure and to demonstratethe practice and advantages thereof. It is understood that the examplesare given by way of illustration and are not intended to limit thespecification or the claims in any manner.

Example 1

Example 1. Extraction of ethyl ferulate and ethyl coumarate from cornfiber. In this example, 1.005 g of corn fiber that was previously driedat 100° C. for 24 hr was loaded into a 100 mL stirred batch reactor(Parr Instruments 2430) with 20 mL 200 proof anhydrous ethanol. Thereactor was sealed and purged with 99.999% hydrogen (Airgas Hy UHP) fourtimes by pressurizing the reactor to 17.2 bar and subsequently ventingthe pressure to ca. 3 bar. The reactor was pressurized to a finalpressure of 34.5 bar. The temperature was increased to 200° C. at a ramprate of 300° C. hr⁻¹ while the reactor was stirred at 200 rpm. Thetemperature was held for 12 hours before returning to room temperature.After reaction, the solvent and solids were filtered through filterpaper (particle retention >11 μm) and rinsed with methanol to a totalvolume of 50 mL. The ethyl ferulate content of the liquid was analyzedby HPLC Method 1, as shown in FIG. 5. Ethyl coumarate and ethyl ferulateeluted at ca. 30.4 and 31.6 min, respectively. The yield of ethylferulate was determined to be 1.0% with respect to the total dry mass ofcorn fiber (assuming 8% moisture content of the biomass, drymass=0.92*1.005 g=0.925 g).

Example 2

Example 2. Extraction of methyl ferulate and methyl coumarate frommiscanthus. In this example, 255.2 g of miscanthus was loaded into a 7.5L stirred batch reactor (Parr Instruments 4550) with 4 L methanol. Thereactor was sealed and purged with 99.999% argon (Airgas Ar UHP) fourtimes by pressurizing the reactor to 7 bar and subsequently venting thepressure to ca. 2 bar. The reactor was pressurized to a final pressureof 7.6 bar. The temperature was increased to 200° C. at a ramp rate of150° C. hr⁻¹ while the reactor was stirred at 200 rpm. The temperaturewas held for 12 hours before returning to room temperature. Afterreaction, the solvent and solids were filtered through a nylon bag(particle retention >75 μm) and rinsed with methanol to a total volumeof 3.2 L. The methyl ferulate and coumarate content of the liquid wasanalyzed by HPLC Method 1, as shown in FIG. 6. Methyl coumarate andmethyl ferulate eluted at ca. 25.5 and 26.5 min, respectively. The yieldof methyl ferulate and methyl coumarate were determined to be 0.5% and3.9%, respectively, with respect to the total dry mass of miscanthus(assuming 8% moisture content of the biomass, dry mass=0.92*255.2g=234.8 g).

Example 3

Example 3. Extraction of ethyl ferulate from corn fiber at 170° C. Inthis example, 7.5 kg of corn fiber previously dried at 90° C. for 24 hrunder vacuum was loaded into a 113.6 L stirred batch reactor with 60 L200 proof anhydrous ethanol and 144 g NaOH. The reactor was sealed andpurged with nitrogen 3 times by pressurizing the reactor to 7 bar andsubsequently venting the pressure to ca. 2 bar. The reactor was filledwith nitrogen to a final pressure of 1 bar. The temperature wasincreased to 170° C. at a ramp rate of 225° C. hr⁻¹ while the reactorwas stirred at 600 rpm. The temperature was held for 12 hours beforecooling to room temperature. After reaction, the solvent and the solidswere filtered through a nylon filter bag and rinsed with 19 L ethanol.The ethanol oil was concentrated with a rotary evaporator to 3.1 kg. Theethyl ferulate content of the oil was analyzed by HPLC Method 1, asshown in FIG. 7. Ethyl coumarate and ethyl ferulate eluted at ca. 31.2and 32.4 min, respectively. The yield of ethyl ferulate was determinedto be 1.5% with respect to the total dry biomass (assumed to be 7.5 kgin this case because the fiber was dried immediately before processing).

Example 4

Example 4. Extraction of ethyl ferulate from corn fiber at 145° C. Inthis example, 1.0 g of corn fiber that was previously dried at 100° C.for 24 hr was loaded into a 100 mL stirred batch reactor (ParrInstruments 2430) with 20 mL 200 proof anhydrous ethanol. To thereaction was added 32 mg NaOH. The reactor was sealed and purged with99.999% argon (Airgas Ar UHP) four times by pressurizing the reactor to17.2 bar and subsequently venting the pressure to ca. 3 bar. The reactorwas pressurized to a final pressure of 3 bar with Argon gas. Thetemperature was increased to 145° C. at a ramp rate of 300° C. hr⁻¹while the reactor was stirred at 200 rpm. The temperature was held at145° C. for 12 hours before returning to room temperature. Afterreaction, the solvent and solids were filtered through filter paper(particle retention >11 μm) and rinsed with methanol to a total volumeof 50 mL. The ethyl ferulate content of the liquid was analyzed by HPLCMethod 2, as shown in FIG. 8. Ethyl coumarate and ethyl ferulate elutedat ca. 30.4 and 31.6 min, respectively. The yield of ethyl ferulate wasdetermined to be 1.66% with respect to the total dry mass of corn fiber(assuming 8% moisture content of the biomass, dry mass=0.92*1.0 g=0.92g).

Example 5

Example 5. Extraction of ethyl ferulate from corn fiber at 120° C. Inthis example, 1.0 g of corn fiber that was previously dried at 100° C.for 24 hr was loaded into a 100 mL stirred batch reactor (ParrInstruments 2430) with 20 mL 200 proof anhydrous ethanol. To thereaction was added 32 mg NaOH. The reactor was sealed and purged with99.999% argon (Airgas Ar UHP) four times by pressurizing the reactor to17.2 bar and subsequently venting the pressure to ca. 3 bar. The reactorwas pressurized to a final pressure of 3 bar with Argon gas. Thetemperature was increased to 120° C. at a ramp rate of 300° C. hr⁻¹while the reactor was stirred at 200 rpm. The temperature was held at120° C. for 12 hours before returning to room temperature. Afterreaction, the solvent and solids were filtered through filter paper(particle retention >11 μm) and rinsed with methanol to a total volumeof 50 mL. The ethyl ferulate content of the liquid was analyzed by HPLCMethod 2, as shown in FIG. 9. Ethyl coumarate and ethyl ferulate elutedat ca. 30.4 and 31.6 min, respectively. The yield of ethyl ferulate wasdetermined to be 1.52% with respect to the total dry mass of corn fiber(assuming 8% moisture content of the biomass, dry mass=0.92*1.0 g=0.92g).

Example 6

Example 6. Extraction of methyl ferulate and methyl coumarate from acontinuous reactor. In this example, 172.3 g of corn fiber previouslydried at 100° C. for 24 hr was loaded into a 1 L packed bed reactorwhich was insulated inside a furnace. The back pressure regulator wasset to 7 bar, and the reactor was filled with methanol at a flow rate of3 mL min⁻¹. The back pressure regulator was increased to 46.9 bar whilethe furnace temperature was ramped such that an internal thermocouple incontact with the corn fiber at the exit of the packed bed recorded atemperature of 200° C., which required 3 hr. The packed bed wasmaintained at 46.9 bar and 200° C. for 6 hr before the furnace wasturned off and the methanol flow rate was increased to 10 mL min⁻¹ for10 min to flush the corn fiber bed. The methanol flow was stopped andthe reactor was cooled to room temperature overnight. Methanol from thesystem was collected in a reservoir with a total volume of 1.81 L. Themethyl ferulate content of the oil was analyzed by HPLC Method 1, asshown in FIG. 10. Methyl coumarate and methyl ferulate eluted at ca.24.2 and 25.3 min, respectively. The yield of methyl ferulate and methylcoumarate was determined to be 1.5% and 1.3%, respectively, with respectto the total dry biomass (assuming 8% moisture content of the biomass,dry mass=0.92*172.3 g=158.5 g). The hourly extraction of methyl ferulateand methyl coumarate is shown in FIG. 11. As shown in FIG. 11, time 0 hrcorresponds to the first time the internal bed temperature at the exitwas 200° C.

Example 7

Example 7. Extraction of methyl ferulate and methyl coumarate from cornbran. In this example, 591 g of corn bran was loaded into a 7.5 Lstirred batch reactor (Parr Instruments 4550) with 4.4 L methanol. Thereactor was sealed and purged with 99.999% argon (Airgas Ar UHP) fourtimes by pressurizing the reactor to 7 bar and subsequently venting thepressure to ca. 2 bar. The reactor was pressurized to a final pressureof 7.6 bar. The temperature was increased to 200° C. at a ramp rate of150° C. hr⁻¹ while the reactor was stirred at 200 rpm. The temperaturewas held for 12 hours before returning to room temperature. Afterreaction, the solvent and solids were filtered through a nylon bag(particle retention >75 μm) and rinsed with methanol to a total volumeof 3.8 L. The methyl ferulate and coumarate content of the liquid wasanalyzed by HPLC Method 1, FIG. 12. Methyl coumarate and methyl ferulateeluted at ca. 20.4 and 22.3 min, respectively. The yield of methylferulate was determined to be 2.3% with respect to the total dry mass ofcorn bran (assuming 8% moisture content of the biomass, drymass=0.92*591 g=543.7 g).

Example 8

Example 8. Hydrolysis of ethyl ferulate to ferulic acid. In thisexample, 19.8 g of concentrated ethanol oil containing ethyl ferulateand ethyl coumarate extracted from corn fiber was added to 50 mLdeionized water with 4 N NaOH. The oil was soluble in aqueous base. Thesolution was stirred in a round bottom and heated to 34° C. for 35 min,which hydrolyzed ethyl ferulate and ethyl coumarate to ferulic acid andcoumaric acid. HPLC chromatograms for the concentrated ethanol oil(Method 1) and the hydrolyzed solution (Method 2) are shown in FIG. 13.No ethyl ferulate is present in the post hydrolysis solution, owing tothe full conversion of ethyl ferulate to ferulic acid and ethylcoumarate to coumaric acid. The results are shown in FIG. 13. Asillustrated, a HPLC/UV-vis chromatogram was recorded at 238 nm. The red,dashed lines show the results from Method 1 applied for crude oilcontaining ethyl ferulate and ethyl coumarate. The blue, solid linesshow the results from Method applied for hydrolyzed crude oil. Ethylcoumarate and ethyl ferulate eluted at ca. 30.7 and 31.9 min,respectively. Coumaric acid and Ferulic acid eluted at ca. 9.8 and 12.2min, respectively.

Example 9

Example 9. Corn oil removal. In this example, 500 g of concentratedethanol oil containing ethyl ferulate and ethyl coumarate extracted fromcorn fiber was added to 1 L deionized water with 0.5 N NaOH. The oil wassoluble in aqueous base. The aqueous solution was placed into aseparatory funnel and washed with 330 mL hexanes. The hexanes was thenremoved from the separatory funnel and the aqueous solution was washedwith 330 mL hexanes two additional times. In total 990 mL hexanes wasused. The hexane extract was then placed in a round bottom flask and thehexanes removed with a rotary evaporator to yield liquid corn oil.Following extraction of the aqueous ethyl ferulate and ethyl coumaratesolution with hexanes, the volume of the aqueous layer was 1.5 L and theethyl ferulate and ethyl coumarate remained in the aqueous layer. HPLCanalysis (Method 2) of the aqueous phase following hexane extractionconfirmed that ethyl ferulate and ethyl coumarate remained in theaqueous layer as shown in FIG. 14. ¹HNMR analysis of the corn oil usinga Varian Unity Inova 400 MHz spectrometer was used to confirm that thecorn oil is comprised of primarily the ethyl esters of oleic andlinoleic acid as shown in FIG. 15.

Example 10

Example 10. Hydrolysis of ferulate to ferulic acid. In this example 1.9L aqueous ethyl ferulate and ethyl coumarate solution from which cornoil had been previously extracted was used. An additional 32 g NaOH wasadded to the aqueous ethyl ferulate and ethyl coumarate solutionincreasing the total concentration of NaOH to 0.76 N. The resultingsolution was heated to 60° C. and held at 60° C. for two hours, whichhydrolyzed ethyl ferulate and ethyl coumarate to ferulic acid andcoumaric acid. Following heating, HPLC analysis (Method 2) was used toconfirm the complete hydrolysis of ethyl ferulate and ethyl coumarate toferulic acid and coumaric acid as shown in FIG. 16.

Example 11

Example 11. Precipitation of lignin and polysaccharides. In this example650 mL of aqueous ferulic acid and coumaric acid solution produced inExample 10 was added to 1.3 L water. The resulting solution was heatedto 90° C. To the heated solution, 10 mL concentrated sulfuric acid wasadded dropwise over a time period of five minutes, acidifying thesolution to pH 4.5. Following addition of the sulfuric acid, a solidprecipitate was visible in the solution. The solution was filtered witha Buchner funnel and filter paper, separating the solid lignin andpolysaccharide mixture from the aqueous ferulic acid and coumaric acidsolution. The volume of the aqueous ferulic acid and coumaric acidsolution was 1.85 L and the mass of the precipitatedlignin/polysaccharide mixture was 20.7 g.

Example 12

Example 12. Extraction of ferulic acid into an organic solvent. In thisexample 1.04 L aqueous ferulic acid and coumaric acid solution fromwhich lignin and polysaccharides had been precipitated, as described inExample 11, was used. The aqueous ferulic acid and coumaric acidsolution was heated to 80° C. and 1.5 mL concentrated sulfuric acid wasadded to the solution. After addition of the sulfuric acid, the pH ofthe solution was reduced to pH 3. The solution was then added to aseparatory funnel and extracted with 330 mL ethyl acetate. Thisextraction was repeated two additional times, with a total of 990 mLethyl acetate used to extract the aqueous solution. Following extractionof the aqueous solution, the ethyl acetate samples were combined andcondensed by rotary evaporation. Rotary evaporation of the ethyl acetatefraction yielded 9.1 grams of a dark oil which contained 2.7 g ferulicacid.

Example 13

Example 13. Crystallization of ferulic acid. In this example, 9.1 gramsof an oil containing 2.7 g ferulic acid was used, this oil was theproduct of Example 12. 100 mL deionized water was added to the ferulicacid containing oil and the solution was heated to 100° C. and theferulic acid was dissolved. The heated solution was filtered using aBuchner funnel and filter paper to remove solid impurities. The filterpaper was rinsed with 20 mL deionized water which had been pre-heated toa temperature of 85° C. The aqueous filtrate was cooled to 5° C. andcrystals of high purity ferulic acid precipitated from the solution overa time period of 12-18 hours. A HPLC chromatogram of ferulic acid with apurity of equal to or greater than 98% purity is shown in FIG. 17.

Having described the various systems and methods, various embodiments asdisclosed herein can include, but are not limited to:

In a first aspect, an integrated process for the reactive separation oforganic molecules from biomass comprises: a reaction step for thebiomass, a simultaneous extraction step using a solvent, and afiltration step to recover the products, wherein the products comprise aferulate or a coumarate.

In a second aspect, a reactive separation process for the separation oforganic molecules including acidic esters, terpenoids, sterols,carbohydrates, and flavonoids from biomass comprises: a reaction stepusing a base in contact with the biomass, a simultaneous solventextraction step using a solvent, and a filtration step to recover theproducts comprising the organic molecules.

A third aspect can include the process of any of the first or secondaspects, wherein the products comprise at least one of: ferulate(ferulic acid ester), and coumarate (coumaric acid ester), wherein theproducts are extracted from the biomass in a pressurized stirred batchreactor using a liquid extraction solvent and the base in which theferulate and the coumarate remain soluble and the insoluble solids arefiltered from the liquid phase and washed.

A fourth aspect can include the process of any of the first to thirdaspects, wherein the reactor contains liquid and a pressurized gasconsisting of nitrogen, argon, helium, or hydrogen, or their mixtures,or wherein the reactor contains a 100% inert atmosphere.

A fifth aspect can include the process of any of the first to fourthaspects, wherein the dry biomass is obtained from agricultural products.

A sixth aspect can include the process of any of the first to fifthaspects, wherein the biomass is mixed with a solvent containing 50-100%any aliphatic alcohols and 0-50% water.

A seventh aspect can include the process of any of the first to sixthaspects, wherein the extraction solvent is 100% ethanol or 100%methanol, or wherein the extraction solvent comprises a biologicallyobtained ethanol.

An eighth aspect can include the process of any of the first to seventhaspects, wherein the mass ratio of solvent to biomass is in the range of4 to 30, or in the range of between 10 and 15.

A ninth aspect can include the process of any of the first to eighthaspects, wherein the base is any first or second group hydroxide,carbonate, bicarbonate, or ammonium hydroxide.

A tenth aspect can include the process of the ninth aspect, wherein thebase is between 0-1 N, or about 0.04 N NaOH.

An eleventh aspect can include the process of any of the first to tenthaspects, wherein the reaction step is carried out in a reactor, andwherein the reactor is pressurized to 1-3 bar at room temperature, orabout 1 bar.

A twelfth aspect can include the process of any of the first to eleventhaspects, wherein the reaction step is carried out in a reactor, andwherein the reactor is heated at 100-300° C. hr⁻¹ to a reactiontemperature of between about 80-250° C., or wherein the heating rate isabout 300° C. hr⁻¹.

A thirteenth aspect can include the process of the twelfth aspect,wherein the reactor is held at the reaction temperature for 1-15 hours,or about 12 hours.

A fourteenth aspect can include the process of any of the first tothirteenth aspects, wherein the reactor is stirred at a rate betweenabout 100-600 rpm, or at about 200 rpm.

A fifteenth aspect can include the process of any of the first tofourteenth aspects, wherein the filtration step produces filteredsolids, and wherein the filtered solids recovered from the reaction arewashed with 50-150% the original volume of the extraction solvent used.

A sixteenth aspect can include the process of any of the first tofifteenth aspects, further comprising: diluting the products with anaqueous solution comprising a base to create a diluted product;contacting the diluted product with an organic solvent; extracting oneor more organic molecules from the diluted product into the organicsolvent to produce a rich organic phase and a purified product.

A seventeenth aspect can include the process of any of the sixteenthaspect, further comprising: removing the organic solvent from the richorganic phase to produce a oil phase and the organic solvent.

An eighteenth aspect can include the process of any of the first toseventeenth aspects, further comprising: combining the products with anaqueous base solution to form a hydrolysis mixture; heating thehydrolysis mixture; and hydrolyzing any ferulates to ferulic acid in thehydrolysis mixture in response to the heating.

A nineteenth aspect can include the process of any of the first toeighteenth aspects, further comprising: adding an acid to the products;acidifying the products in response to adding the acid to produceacidified products; precipitating one or more byproducts from theacidified products; and removing the one or more byproducts as solidsfrom the acidified products, wherein a concentration of ferulic acid inthe acidified products is greater after removing the one or morebyproducts than prior to acidifying the products.

A twentieth aspect can include the process of the nineteenth aspect,further comprising: contacting the acidified products with an organicsolvent after removing the one or more byproducts; extracting theferulic acid from the acidified products using the organic solvent toform a rich organic solvent; removing the organic solvent from the richorganic solvent; and producing a solid ferulic acid in response toremoving the organic solvent from the rich organic solvent.

A twenty first aspect can include the process of the twentieth aspect,further comprising: dissolving the solid ferulic acid in an aqueoussolution to form dissolved ferulic acid; heating the dissolved ferulicacid; filtering the dissolved ferulic acid; removing one or moreinsoluble impurities from the dissolved ferulic acid using thefiltering; and cooling the dissolved ferulic acid after removing the oneor more insoluble impurities to produce purified solid ferulic acid.

A twenty second aspect can include the process of any of the twentiethor twenty first aspects, further comprising: purifying the ferulic acidusing liquid chromotography.

In a twenty third aspect, a process to extract ferulate (ferulic acidester) and coumarate (coumaric acid ester) from agricultural biomass ina packed bed reactor, wherein the process comprises: contacting thebiomass with a solvent and a base in the packed bed reactor, wherein theagricultural biomass acts as the stationary bed.

A twenty fourth aspect can include the process of the twenty thirdaspect, wherein the solvent comprises an aliphatic alcohol, and thesolvent is pumped through the reactor at a flow rate of between 1-10 mLmin⁻¹, or at a flow rate between about 1.5-3 mL min⁻¹.

A twenty fifth aspect can include the process of the twenty third ortwenty fourth aspect, further comprising contacting the biomass with abase in the packed bed reactor with the solvent, wherein the base is anyfirst or second group hydroxide, carbonate, bicarbonate, or ammoniumhydroxide and has a concentration between 0-1 N, or about 0.04 N.

A twenty sixth aspect can include the process of any of the twenty thirdto twenty fifth aspects, wherein the packed bed reactor is pressurizedto between about 13-30 bar, or to between about 13-20 bar.

A twenty seventh aspect can include the process of any of the twentythird to twenty sixth aspects, wherein the temperature of the packed bedreactor is heated at a rate of between about 100-300° C. hr⁻¹, or at arate of about 300° C. hr⁻¹, to a desired temperature of between about80-250° C.

A twenty eighth aspect can include the process of the twenty seventhaspect, wherein the reactor is held at the desired reactor temperaturefor 4-8 hours.

A twenty ninth aspect can include the process of any of the twenty thirdto twenty eighth aspects, wherein after an appropriate dwell time thereactor is cooled 20° C., and the solvent flow rate is increased to 10mL min⁻¹ to flush the reactor zone into the collection container.

A thirtieth aspect can include the process of the first, second, ortwenty third aspects in which the solids from the products are filteredfrom the liquid phase, and then the solvent is concentrated to a viscousoil using a rotary evaporator.

In a thirty first aspect, a process in which ferulate and coumaratecontaining oil, such as any oil obtained in one of the first tothirtieth aspects is suspended in water and extracted with hexanesfollowed by ethyl acetate, leaving the ferulate and coumarate in theethyl acetate phase.

A thirty second aspect can include the process of the thirty firstaspects in which the ethyl acetate phase is distilled in a short pathdistillation apparatus such as a Kugelrohr or wiped thin film evaporatorat fractions up to 200° C.

In a thirty third aspect, a process in which the ferulate and coumarate,for example as obtained in any of the first to thirty second aspects,are hydrolyzed to ferulic acid under basic conditions.

A thirty fourth aspect can include the process of the thirty thirdaspect, wherein 2-20 mL of water are added per gram ferulate products.

A thirty fifth aspect can include the process of the thirty third orthirty fourth aspect, wherein the base includes any first or secondgroup hydroxides such as sodium hydroxide or potassium hydroxide,carbonates, bicarbonates, or ammonium in a concentration of 0.1-10 N.

A thirty sixth aspect can include the process of any of the thirty thirdto thirty fifth aspects, wherein the solution is heated to 30-100° C.

A thirty seventh aspect can include the process of any of the thirtythird to thirty sixth aspects, wherein the hydrolysis is carried out for0.5-5 hours for complete conversion of ferulate to ferulic acid, andcoumarate is converted to coumaric acid.

In the preceding discussion and in the claims, the terms “including” and“comprising” are used in an open-ended fashion, and thus should beinterpreted to mean “including, but not limited to . . . ”. At least oneembodiment is disclosed and variations, combinations, and/ormodifications of the embodiment(s) and/or features of the embodiment(s)made by a person having ordinary skill in the art are within the scopeof the disclosure. Alternative embodiments that result from combining,integrating, and/or omitting features of the embodiment(s) are alsowithin the scope of the disclosure. Where numerical ranges orlimitations are expressly stated, such express ranges or limitationsshould be understood to include iterative ranges or limitations of likemagnitude falling within the expressly stated ranges or limitations(e.g., from about 1 to about 10 includes, 2, 3, 4, etc.; greater than0.10 includes 0.11, 0.12, 0.13, etc.). For example, whenever a numericalrange with a lower limit, Rl, and an upper limit, Ru, is disclosed, anynumber falling within the range is specifically disclosed. Inparticular, the following numbers within the range are specificallydisclosed: R=Rl+k*(Ru−Rl), wherein k is a variable ranging from 1percent to 100 percent with a 1 percent increment, i.e., k is 1 percent,2 percent, 3 percent, 4 percent, 5 percent, . . . , 50 percent, 51percent, 52 percent, . . . , 95 percent, 96 percent, 97 percent, 98percent, 99 percent, or 100 percent. Moreover, any numerical rangedefined by two R numbers as defined in the above is also specificallydisclosed. Use of the term “optionally” with respect to any element of aclaim means that the element is required, or alternatively, the elementis not required, both alternatives being within the scope of the claim.Use of broader terms such as comprises, includes, and having should beunderstood to provide support for narrower terms such as consisting of,consisting essentially of, and comprised substantially of. Accordingly,the scope of protection is not limited by the description set out abovebut is defined by the claims that follow, that scope including allequivalents of the subject matter of the claims. Each and every claim isincorporated as further disclosure into the specification and the claimsare embodiment(s) of the present invention.

What is claimed is:
 1. A process for a reactive separation of organicmolecules from biomass comprising: contacting biomass with a base and asolvent; extracting products from the biomass with the solvent based ona reaction of the base with the biomass to form a slurry; filtering theslurry to recover a liquid product, wherein the liquid productcomprises: a) a ferulate, a coumarate, ferulic acid, coumaric acid, orany combination thereof, and b) a fatty acid compound comprising a fattyacid, a fatty acid ester, or any combination thereof; and removing atleast a portion of the fatty acid compound from the liquid product toproduce a purified liquid product comprising the coumarate, theferulate, the ferulic acid, the coumaric acid, or any combinationthereof.
 2. The process of claim 1, wherein removing at least theportion of the fatty acid compound from the liquid product comprisesremoving at least the portion of the fatty acid compound from the liquidproduct by a liquid-liquid extraction to produce the purified liquidproduct.
 3. The process of claim 1, wherein removing at least theportion of the fatty acid compound from the liquid product comprisesremoving the fatty acid compound from the liquid product by liquidchromatography to produce the purified liquid product comprising thecoumarate, the ferulate, the ferulic acid, the coumaric acid, or anycombination thereof.
 4. The process of claim 1, wherein removing atleast the portion of the fatty acid compound from the liquid productcomprises removing the fatty acid compound from the liquid product byion exchange chromatography or size exclusion chromatography to producethe purified liquid product comprising the coumarate, the ferulate, theferulic acid, the coumaric acid, or any combination thereof.
 5. Theprocess of claim 1, wherein removing at least the portion of the fattyacid compound from the liquid product comprises removing the fatty acidcompound from the liquid product as an insoluble impurity by dissolvingthe ferulic acid in an aqueous solution to form the insoluble impurityand removing the insoluble impurity by filtration or phase separation.6. The process of claim 1, wherein removing at least the portion of thefatty acid ester from the liquid product comprises hydrolyzing theliquid product under basic conditions to convert at least the portion ofthe fatty acid ester to a corresponding acid, wherein the liquid productcomprises organic molecules, wherein the organic molecules compriseacidic esters, terpenoids, sterols, carbohydrates, and flavonoids. 7.The process of claim 6, wherein hydrolyzing the liquid product underbasic conditions comprises: combining the liquid product with an aqueousbase solution to form a hydrolysis mixture; heating the hydrolysismixture; and hydrolyzing any ferulates to ferulic acid in the hydrolysismixture in response to the heating.
 8. The process of claim 1, furthercomprising: adding an acid to the liquid product after removing thefatty acid compound; acidifying the liquid product in response to addingthe acid to produce acidified products; precipitating one or morebyproducts from the acidified products; and removing the one or morebyproducts as solids from the acidified products, wherein aconcentration of ferulic acid in the acidified products is greater afterremoving the one or more byproducts than prior to acidifying theproducts.
 9. The process of claim 8, further comprising: contacting theacidified products with an organic solvent after removing the one ormore byproducts; extracting the ferulic acid from the acidified productsusing the organic solvent to form a rich organic solvent; removing theorganic solvent from the rich organic solvent; and producing a solidferulic acid in response to removing the organic solvent from the richorganic solvent.
 10. The process of claim 9, further comprising:dissolving the solid ferulic acid in a heated aqueous solution to formdissolved ferulic acid; filtering the dissolved ferulic acid; removingone or more insoluble impurities from the dissolved ferulic acid usingthe filtering; and cooling the dissolved ferulic acid after removing theone or more insoluble impurities to produce purified solid ferulic acid.11. The process of claim 10, further comprising: purifying the ferulicacid using liquid chromatography.
 12. The process of claim 9, furthercomprising: purifying the ferulic acid using liquid chromatography. 13.The process of claim 1, wherein removing the fatty acid compound fromthe liquid product comprises: generating a rich organic phase and thepurified liquid product, wherein the rich organic phase comprises thefatty acid compound and the solvent.
 14. The process of claim 13,further comprising: removing the solvent from the rich organic phase toproduce an oil phase and the solvent, wherein the oil phase has aphosphorus content of less than 250 ppm by mass.
 15. The process ofclaim 14, wherein the oil phase has an oleic acid content of less than3% by mass of the oil phase.
 16. The process of claim 1, whereincontacting the biomass with the base and the solvent occurs in thepresence of water, wherein the water is present in a mass ratio of thewater to the biomass of less than 15:1.
 17. The process of claim 1,wherein the liquid product is extracted from the biomass in a reactorcomprising a pressurized stirred batch reactor or a stirred batchreactor, and wherein the reactor contains liquid and a gas consisting ofnitrogen, argon, helium, or hydrogen, or their mixtures.
 18. The processof claim 1, wherein the solvent comprises 50-100% any aliphatic alcoholsand 0-50% water, and wherein a mass ratio of the solvent to the biomassis in a range of 4:1 to 30:1.
 19. The process of claim 1, wherein thereaction step is carried out in a reactor, wherein the reactor is heatedto a reaction temperature of between about 80-250° C., and wherein thereactor is held at the reaction temperature for 1-15 hours.
 20. Theprocess of claim 1, wherein the solvent comprises 50-100% any aliphaticalcohols and 0-50% water.